Due to its central role in both evolutionary change and human disease, mutation has been the focus of intensive research. The probability that a spontaneous mutation will occur and its impact on fitness are parameters of central importance for making predictions in evolutionary biology. However, estimates of these parameters are difficult to make, and exist only for a handful of taxonomically-restricted eukaryotic species. Here we introduce a new experimental system for elucidating these mutational parameters: the ciliate Tetrahymena thermophila. T. thermophila has some unusual features that make it particularly well-suited to studying spontaneous mutations. Crucially, the sequestration of a transcriptionally-inactive germline genome, within the same cell as a somatic genome, means that mutations in the germline can be sheltered from selection over the course of thousands of cell divisions. We propose to use T. thermophila in a mutation accumulation experiment. Follow-up experiments with the lines that we generate using a range of complementary genetic and genomic tools, combined with powerful computational and statistical analyses will allow us to estimate germline mutational parameters with high robustness and accuracy, including: the overall mutation rate; the rate and effect sizes of mutations deleterious to fitness; their dominance and epistatic relationships; the rate of recessive lethal mutations; and the spectrum of mutations at the molecular level.